The human inducible nitric oxide synthase (hiNOS) gene is expressed in nearly every organ during sepsis and other inflammatory conditions. While NO synthesis has beneficial effects during acute inflammation, excessive NO production is harmful. Chronic hiNOS expression has been implicated in NO-mediated tissue damage leading to diabetes, neurodegenerative disorders, and certain cancers. Our laboratory has cloned the human iNOS gene from eytokine-stimulated hepatoeytes, and we have shown that cytokine-responsive DNA elements are located approximately 5 kb upstream in the promoter region. We found that TNFalpha and IL-1beta signal through NF-kappaB, while IFNgamma signals through Stat-1 by binding to cis-acting elements at -5.2 and -5.8 kb in the promoter, thereby providing a molecular basis for cytokine synergy. Further functional roles have been demonstrated for AP-1, C/EBPbeta (LAP), and KLF6. Importantly, we have identified mechanisms for hiNOS repression that involve NF-kappaB repressing factor (NRF), LIP, and p53 proteins. A subsequent chromatin structure analysis using DNAse I mapping and in vivo footprinting revealed that regulation of hiNOS transcription was even more complex than originally anticipated and exhibited tissue-specific control by both basal and inducible transcription factors. Most recently, we have identified a novel role for the Wnt beta-catenin/Tcf-4 signaling pathway in regulating hiNOS expression. Therefore, our hypothesis is that the regulation of hiNOS gene expression requires an orchestrated flow of positive and negative transcription factors binding to a cis-acting upstream enhancer region located between -5.0 and -7.0 kb in the hiNOS promoter. In addition, a crucial downstream promoter region has been identified at -0.2 kb that is permissive for cytokine-induced transcription. In this proposal, we will pursue two specific aims to further elucidate the molecular mechanisms involved: AIM I: TO DEFINE THE TRANSCRIPTION FACTORS AND FUNCTIONAL PROMOTER ELEMENTS RESPONSIBLE FOR CYTOKINE INDUCTION OF THE HUMAN iNOS GENE. ChIP assay will be used to confirm in vivo protein-DNA interactions for NF-kappaB, Stat-1, and AP-1 in the upstream enhancer region. New roles for Ets-1 and Oct-1 will be tested by gel shift and promoter transfection studies. Protein-protein interactions between NRF and NF-kB will be pursued, as well as mechanisms of downstream control elicited by LAP/LIP, KLF6, and p53. AIM II: TO DETERMINE THE ROLE OF THE WNT Beta-CATENIN/TCF-4 SIGNALING PATHWAY IN REGULATING HUMAN iNOS EXPRESSION. A functional role for Beta-catenin/Tcf-4 binding to two TBE sites will be determined, as well as possible interactions with other transcription factors governing hiNOS expression. The information gained will increase our understanding of the control of hiNOS transcription, describe novel mechanisms of cytokine-synergy in signal transduetion, and help in designing therapeutic strategies for pathophysiologic disease states where hiNOS expression is relevant.